Chromatographic separation of phosphorothioate oligonucleotides

ABSTRACT

A method is provided for the preparation and/or analysis of synthetic phosphorothioate and -dithioate oligonucleotides. In particular, the method permits separation of fully sulfurized phosphorothioate or -dithioate oligonucleotides from incompletely sulfurized defect species on an anion exchange HPLC columns using concentration gradients of novel &#34;soft base&#34; anionic eluents, such as bromide, thiocyanate, and the like.

This is a continuation-in-part application of U.S. patent applicationSer. No. 07/779,012, filed Oct. 18, 1991, now U.S. Pat. No. 5,183,885.

FIELD OF THE INVENTION

The invention relates generally to the preparation and/or analysis ofsynthetic phosphorothioate and -dithioate oligonucleotides, and moreparticularly, to a method for separating fully sulfurizingphosphorothioate or -dithioate oligonucleotides from incompletelysulfurized defect species by strong-anion exchange high performanceliquid chromatography (HPLC).

BACKGROUND

Anti-sense oligonucleotides are being developed to treat a variety ofdiseases, particularly vital infections, e.g. Matsukura et al, Proc.Natl. Acad. Sci., Vol. 86, pgs. 4244-4448 (1989). An antisenseoligonucleotide is a synthetic oligonucleotide of varying length,usually in the range of about 12 to 30 nucleotides, or nucleotideanalogs, whose sequence is complementary to a predetermined segment ofRNA, either freshly transcribed or messenger (mRNA), associated withsome foreign or otherwise inappropriately expressed gene. It is believedthat when an antisense oligonucleotide hybridizes to its target RNA, iteither blocks translation or processing of the RNA or makes itsusceptible to enzymatic degradation. One problem with this approach hasbeen the difficulty of getting the antisense oligonucleotide to itstarget RNA in sufficient concentration and for sufficient duration to beeffective in shutting down the synthesis of undesired proteins, e.g.viral enzymes, coat proteins, and the like. The susceptibility of thephosphodiester linkage of the oligonucleotides to nuclease digestion isbelieved to be an important cause of this difficulty, and has promptedthe development of a variety of nucleoside oligomers linked bynuclease-resistant analogs of the natural phosphodiester bond, e.g.Miller et al, U.S. Pat. No. 4,511,713 and Ts'o U.S. Pat. No. 4,469,863(methyl- and arylphosphonates); Miro et al, Nucleic Acids Research, Vol.17, pgs. 8207-8219 (1989) (phosphoroselenoates); Brill et al, J. Am.Chem. Soc., Vol. 111, pg. 2321 (1989) (phosphorodithioates); andMatsukura et al, Proc. Natl. Acad. Sci., Vol. 84, pgs. 7706-7710 (1987),and Gene, Vol. 72, pgs. 343-347 (1988) (phosphorothioates).

The phosphorothioate and phosphorodithioate analogs are especiallypromising because they are highly nuclease-resistant, have the samecharge as natural oligonucleotides, and are taken up by cells ineffective amounts.

Phosphorothioates can be synthesized by automated DNA synthesizers usinghydrogen phosphonate or phosphoramidite chemistries. In the formerapproach, the phosphonate backbone can be sulfurized in a single stepoff of the automated synthesizer after synthesis. This is advantageousbecause the phosphonate moieties are sulfurized by exposure to elementalsulfur dissolved in an organic solvent. Since the sulfur readilyprecipitates out of solution, the off-column sulfurization avoids costlyblockages of valves and tubing of the synthesizer by sulfurprecipitates. A drawback of of this route of phosphorothioate synthesisis that coupling yields during chain elongation are typically lower thanthose obtained using phosphoramidite chemistry, Gaffney and Jones, Tett.Lett., Vol. 29, pgs. 2619-2622 (1988). The practical importance of highcoupling yields is demonstrated by the synthesis of a 28-mer where a 99%coupling yield per step results in an overall yield of 76% (0.99²⁷),whereas a 96% yield per step results in an overall yield of only 33%(0.96²⁷).

Phosphoramidite chemistry, with coupling yields typically greater than99%, is presently the more desirable approach to phosphorothioate andphosphorodithioate synthesis. However, the phosphite intermediates,which would be sulfurized, are unstable under the conditions of thedetritylization step of the reaction cycle. Thus, the phosphite linkagemust be sulfurized after each coupling step. This can be accomplishedwith a variety of sulfurizing agents, e.g. Matsukura et al, Gene (citedabove)(elemental sulfur); lyer et al, J. Org. Chem., Vol. 55, pgs.4693-4699 (1990)(a thiosulfonate sulfurizing agent); Hirschbein, PCTapplication US91/01008 (thiuram disulfide and polysulfide sulfurizingagents); and Stec et al, U.S. Pat. No. 5,151,510 (thiophosphorussulfurizing agents). Unfortunately, none of these agents provides 100%sulfurization. At each sulfurization step a small fraction of thephosphite precursors are oxygenated instead of sulfurized. This leads tothe synthesis of a complex mixture of phosphorothioate oligonucleotideswith respect to the number and distribution of oxygens along thephosphodiester backbone. The fraction of a product containing a givennumber of oxygens follows the binomial distribution. For example, in thesynthesis of a 20-mer phosphorothioate oligonucleotide where thesulfurization yield is 99% at each step, the fraction of the productwith, say, 1 and 2 oxygenations in place of sulfurizations is given bythe second and third terms, respectively, of the binomial expansion of##EQU1## respectively. Thus, relatively large fractions of even modestlysized phosphorothioate oligonucleotides are incompletely sulfurized and,because of physiochemical similarity of the completely and incompletelysulfurized compounds, separation and/or analysis of the two species hasproven to be inconvenient, usually requiring NMR analysis, or likeprocedures.

In view of the desire to employ phosphorothioate and phosphorodithioateanalogs of oligonucleotides as pharmaceutical compounds, it would beadvantageous to have available methods for preparation and analysis ofthe sulfurized products that would permit separation of fully sulfurizedspecies from partially sulfurized species and that would permit aconvenient and inexpensive way of monitoring yields of completelysulfurized analogs, particularly in connection with phosphoramiditeand/or phosphorthioamidite chemistries.

DISCLOSURE OF THE INVENTION

The invention is directed to a method for separating fully sulfurizedphosphorothioate and/or -dithioate oligonucleotides from partiallysulfurized species by anion exchange high performance liquidchromatography (HPLC). The method comprises the steps of (1)impregnating an anion exchanger with a mixture of completely sulfurizedand incompletely sulfurized phosphorothioate or phosphorodithioateoligonucleotides, (2) passing a buffer solution through the anionexchanger wherein the buffer solution comprises a concentration of asoft-base counter ion which monotonically increases with time from astarling concentration to a sample-desorbing concentration; and (3)recovering the eluate from step (2) containing the completely sulfurizedphosphorothioate or phosphorodithioate oligonucleotide. The inventionalso forms the basis for a rapid and convenient method for analyzing thedegree of sulfurization of phosphorothioate and/or phosphorodithioateoligonucleotides which uses far less sample than other currentlyavailable techniques.

As used herein "soft-base counter ion" means a base suitable fordisplacing a phosphorothioate moiety from a cationic group on an anionexchanger, and more particularly, it refers to a counter ionic basewhich is characterized by low electronegativity and high polarizabilityand which is chemically stable in the reagents and pH of the buffersolution. Preferably, the soft-base counter ion has negligibleabsorbance in the range of 250-280 nm so that it permits the convenientdetection of the separated oligonucleotides by UV absorption. Guidancefor selecting soft bases is given by Pearson, in J. Amer. Chem. Soc.,Vol. 85, pgs. 3533-3539 (1963); Pearson, J. Chem. Ed., Vol. 45, pgs.581-587 and 643-648 (1968); Pearson et al, J. Amer. Chem. Soc., Vol. 89,pgs. 1827-1836 (1967); and Ho, Chem. Rev., Vol. 75, pgs. 1-20 (1975).Exemplary soft-base counter ions include bromide, thiocyanate, iodide,azide, cyanate, thiosulfate, inorganic sulfide, and organic sulfides ofthe general form RS⁻, wherein R is aryl having 6 to 10 carbon atoms oralkyl having from 1 to 6 carbon atoms. More preferably, R is phenyl,methyl, or ethyl. More preferably, the soft base is selected from thegroup consisting of bromide and thiocyanate.

As used herein the term "sample-desorbing concentration" in reference tothe softbase counter ion means a concentration of a soft-base counterion that is sufficient to elute a fully sulfurized phosphorothioate or-dithioate oligonucleotide from a anion exchanger. This concentrationdepends on the type of soft-base counter ion, the type of the cationicgroup on the exchanger, the organic modifier employed, pH, the length ofthe phosphorothioate or -dithioate oligonucleotides in the sample, andother similar parameters. The sample-desorbing concentration is readilydetermined for particular for particular embodiments by routineexperimentation.

As used herein the term "starting concentration" in reference to thesoft-base counter ion means a concentration of a soft-base counter ionthat ranges from 0.0 to 0.6M at the start of a gradient, and morepreferably, a concentration that ranges from 0.3 to 0.5M at the start ofa gradient.

The term "oligonucleotide" as used herein includes linear oligomers ofnatural or modified nucleosides or of non-nucleosidic analogs linked byphosphodiester bonds or analogs thereof ranging in size from a fewmonomeric units, e.g. 2-3, to several several tens of monomeric units,e.g. 30-50. In particular, the term includes non-natural oligomershaving phosphorous-containing linkages whose phosphorous(III) precursorsare amenable to sulfurization, e.g. Takeshita et al, J. Biol. Chem.,Vol. 282, pgs. 10171-10179 (1987); and Eapienis et al, pgs. 225-230 in,Bruzik and Stec, eds., Biophosphates and Their Analogs--Synthesis,Structure, Metabolism, and Activity (Elsevier, Amsterdam, 1986).

As used herein, "incompletely sulfurized" in reference tophosphorothioate or -dithioate oligonucleotide means that one or more ofthe non-bridging oxygens of one or more of the phosphodiester linkageshave failed to be replaced with sulfur. Conversely, "completelysulfurized" in reference to phosphorothioate or -dithioateoligonucleotide means that one (-thioate) or both (-dithioate) of thenon-bridging oxygens of every phosphodiester linkage have been replacedwith sulfur.

Mixtures of completely sulfurized and incompletely sulfurizedphosphorothioate or phosphorodithioate oligonucleotides for use in themethod of the invention can arise from all currently available methodsof synthesizing phosphorothioate and phosphorodithioateoligonucleotides. Detailed procedures for the phosphoramidite,phosphorthioamidite, and hydrogen phosphonate methods of oligonucleotidesynthesis are described in the following references, which areincorporated by reference: Caruthers et al, U.S. Pat. Nos. 4,458,066 and4,500,707; Koester et al, U.S. Pat. No. 4,725,677; Matteucci et al, J.Amer. Chem. Soc., Vol. 103, pgs. 3185-3191 (1981); Caruthers et al,Genetic Engineering, Vol. 4, pgs. 1-17 (1981); Jones, chapter2, andAtkinson et al, chapter 3, in Gait, ed., Olionucleotide Synthesis: APractical Approach (IRL Press, Washington, D.C., 1984); Froehler et al,Tetrahedron Letters, Vol. 27, Pgs. 469-472 (1986); Garegg et al,Tetrahedron Letters, Vol. 27, pgs. 4051-4054 and 4055-4058 (1986);Andrus et al, U.S. Pat. No. 4,816,571; Brill et al, J. Am. Chem. Soc.,Vol. 111, pgs. 2321- (1989); Caruthers et al, PCT applicationUS89/02293; Stec et al, European application No. 92301950.9; andFroehler et al, Nucleic Acids Research, Vol. 14, pgs. 5399-5407 (1986).Various sulfurization methods are disclosed by Matsukura et al, Gene(cited above); lyer et al, J. Org. Chem., Vol. 55, pgs. 693-4699 (1990);Hirschbeth, PCT application No. US91/01008; Beaucage et al, U.S. Pat.No. 5,003,097; and Stec et al, U.S. Pat. No. 5,151,510.

Preferably, the methods of the invention are implement by highperformance, or high pressure, liquid chromatography (HPLC). Extensiveguidance for choosing particular design parameters, e.g. column size,flow rates, anion exchanger (both matrix and covalently attachedcationic group), and the like, is available in any of many texts onliquid chromatography, e.g. Snyder and Kirkland, Introduction to ModernLiquid Chromatography, 2nd Ed. (Wiley Interscience, New York, 1979).

A variety of anion exchangers can be used, but preferably the exchangermatrix or resin must be suitable for use in HPLC. For example, it mustbe mechanically rigid, stable under ordinary operating pressures, inertwith respect to the solvents employed, stable in the pH range employed,and the like. Preferably, anion exchanger matrices include agarose,silica gel, methacrylic polymers, and highly cross-linkedstyrene-divinylbenzene matrices, with the latter being most preferred.Preferably the matrix is derivatized with cationic ion-exchangefunctionalities, particularly ternary and quaternary alkylammoniumcations, such as triethylaminoethyl, diethylaminoethyl,diethyl-(2-hydroxypropyl)aminoethyl, quaternary aminoethyl, quaternaryamine, or the like. Preferably, the matrix is derivatized withquaternary amine.

The particle size is an important factor in determining how well thecompletely and incompletely phosphorothioate oligonucleotides areresolved as separate peaks in the chromatographic process. Preferably,the particle size is less than about 15 μm diameter, and morepreferably, particle size is in the range of about 8-10 μm. The porosityof the ion exchange resin is not critical. Pore size in the range of 60to 1000 angstroms is suitable.

The temperature considerations of the process are similar to those ofany ion exchange process. The appropriate operating temperature willthus depend on the volume of the exchanger in the column, the particlesize, the surface area and other similar variables and can be readilydetermined by routine experimentation. It will be most convenient tooperate at a temperature within the range of about 14° C. to about 35°C., preferably from about 17° C. to about 30° C., and most preferably,from about 20° C. to about 28° C.

The buffer solution is a liquid medium in which counter ions aredelivered to the adsorbed sample at a controlled concentration and pH.Optionally, the buffer solution may contain one or more organicmodifiers to assist in the desorption process. Preferably, organicmodifiers are included in the buffer solution (1) to ensure that theoligonucleotides in the sample remain single stranded, and (2) toneutralize hydrophobic interactions between the oligonucleotides andsuppod matrix. In further preference, to avoid interference with thedetection of the eluted oligonucleotides, the organic modifier should betransparent to light in the UV wavelength range, e.g. 250-280 nm.Preferable organic modifiers include acetonitrile, formamide, urea, andlike solvents. Most preferably, acetonitrile is used as an organicmodifier.

The pH may be optimized for a particular soft-base counter ion.Likewise, the concentration gradient of the soft-base counter ion,including functional shape, time course, and the like, are selected tooptimize separation and can be readily determined by routineexperimentation taking into account column size, flow rates, amount ofadsorbed sample, and the like. In some cases, the selection of anionexchanger matrix can affect selection of pH ranges. For example, silicagel matrices are confined to buffers having pH in the range of about 2-7due to dissolution of the support under alkaline conditions, e.g.pH>7.5. Generally, buffer pH is not a critical variable and can rangefrom 6-14. Preferably, the concentration of the soft-base counter ion isincreased monotonically until the sample desorbing concentration isreached, after which the concentration is held substantially constantfor a time long enough to remove substantially all the completelysulfurized phosphorothioate oligonucleotide. More preferably, theconcentration of the soft-base counter ion increases linearly, and mostpreferably, at a rate in the range of about 1-2 percent per minute.After elution, the separation column can be recycled by returning theconcentration to the starting value.

The volume and flow rate of the buffer solution to be passed through theexchanger bed will be selected to provide the optimum separation and, asbefore, can be determined by routine experimentation.

When the method of the invention is used analytically, fractions of thecompounds having full or partial sulfurization are readily determined bycomparing chromatogram peak areas with the total area under all peaks.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-e are chromatograms of phosphorothioate oligonucleotidesshowing the separation of components with full sulfurization fromcomponents with one or more unsulfurized phosphodiester linkages. Theanion exchanger was styrene-divinylbenzene copolymer derviatized with aquaternary ammonium cation.

FIG. 2 is a chromatogram of phosphorothioate oligonucleotides showingthe separation of components with full sulfurization from componentswith one or more unsulfurized phosphodiester linkages. The anionexchanger was silica gel derivatized with a diethylaminoethyl ternaryamine cation.

FIG. 3 is a chromatogram showing separation of fully thioated andpartially thioated oligonucleotides under conditions of high pH and noorganic modifier when an exchanger matrix is styrene-divinylbenzenecopolymer.

EXAMPLE 1

In this example four phosphorothioate oligonucleotides were synthesizedand completely sulfurized and incompletely sulfurized components wereseparated in accordance with the invention. The fractions of completelysulfurized and incompletely sulfurized components were confirmed by NMRanalysis. The following phosphorothioate oligonucleotides weresynthesized via phosphoramidite chemistry on either an AppliedBiosystems Model 380B or 390Z automated DNA Synthesizer using standardprotocols, e.g. citations above and Efcavitch, pg. 221 in Schlesinger,ed. Macromolecular Sequencing and Synthesis: Selected Methods andApplications (Alan R. Liss, New York, 1988): S-d(C₁₂ G₅ T₁₀) (1), S-d(A₃C₇ G₇ T₄) (2), S-d(A₁ C₈ G₂ T₉)(3), and S-d(C₁₃ G₃ T₄) (4), wherein (forexample) d(C₁₂ G₅ T₁₀) represents a 27-mer oligodeoxyribonucleotide5'-CCCCCCCCCCCCGGGGGTTTTTTTTTT and the prefix "S-" indicates that thephosphodiester linkages have been sulfurized. Sulfurization steps werecarried out with 3H-1,2-benzothiol-3one 1,1-dioxide, as described bylyer et al (cited above). The 5'-O-dimethoxytrityl derivatives of thecrude DNA from each synthesis was initially purified by reversed-phasepreparative chromatography as described by Zon el al, Biochromatography,Vol. 1, pg. 22 (1988).

Analytical separation of the completely sulfurized and incompletelysulfurized phosphorothioate oligonucleotides was carried out on aPerkin-Elmer Series 410 B10 liquid chromatography system using aPerkin-Elmer ISS-200 Autosampler, a Perkin-Elmer Model 1020 Data System,and an Applied Biosystems, Inc. Model 759A UV detector. The column was15 cm×7.5 mm i.d. loaded with a quaternary alkylammonium functionalizedsuppod, Polymer Labs PL-SAX, with particle size of 10 μm and pore sizeof 1000 angstroms. The buffer solution consisted of three components: A(50 mM ammonium phosphate buffer pH 8.2 and acetonitrile in a 95:5 ratio(v/v)); B (1.5M potassium bromide in 50 mM ammonium phosphate buffer pH6.7 and acetonitrile in an 80:20 ratio (v/v)); and C (acetonitrile). Theconcentration of the bromide soft-base counter ion was brought linearlyfrom 0.45M to 1.2M in 48 minutes by going from 50A:30B:20C to0A:80B:20C. The flow rate was 1.5 ml/min and the eluted material wasdetected by UV absorbance at 260 nm. FIGS. 1a-e are the resultingchromatograms of compound 1,2 (high O), 2 (low O), 3, and 4respectively. The roman numerals above the peaks correspond to the romannumerals in the Tables below.

Peaks I, II, and III of compounds 1 and 2 were preparatively isolatedand o examined by NMR. The preparative isolation was carded out bydissolving about 15 mg of DNA (prepared as described above) in 2 ml ofwater and transferring to the same column as described above via aloop-injector (Rheodyne 9125). The buffer solution and gradient were thesame as above, except that the gradient cycle was 70 min. Fractionsconsisting of 2-12 mg of each peak were collected, individually desired,and taken up in D₂ O for ³¹ P-NMR analysis. NMR analysis was carried outon either a JEOL GSX-500 with sampling frequency of 202.45 MHz,acquisition time of 0.655 s, pulse delay of 6 s, and pulse width of 5 us(45°) or a Varian Unity 3000 with sampling frequency of 121.42 MHz,acquisition time of 1-6 s, pulse delay of 0 s, and pulse width of 111 μs(90° ). Table I compares the NMR-determined percentage of unsulfurizedphosphodiester linkages in the components corresponding to peaks I-IV ofcompounds 1 and 2. It is readily seen that the theoretical values(obtained from the binomial distribution) and the measured values arevery close.

                  TABLE I                                                         ______________________________________                                        Percentage of unsulfurized phosphodiester linkages                            In peaks I-IV of compounds 1 and 2                                                    COMPOUND                                                                      Found by NMR     Theoretical                                          Peak      1      2           1    2                                           ______________________________________                                        I         0.0    0.1         0.0  0.0                                         II        5.1    3.6         3.8  5.0                                         III       8.8    7.7         7.7  10.0                                        IV        N.D.   N.D.        11.5 15.0                                        ______________________________________                                    

Finally, the fractions of 0-, 1-, 2-, and 3-fold oxygenated componentsof compounds 1-4 were determined by the method of the invention andcompared to theoretical fractions(calculated from the binomialdistribution) and the total percent P=S as determined by NMR analysis.The results are displayed in Table II. The multiple entries for the samecompound represent separate syntheses of the same compound, which insome cases were sulfurized less efficiently than others.

                                      TABLE II                                    __________________________________________________________________________    Total Percentage P = S by NMR and percentage of 0-, 1-, 2-, and 3-fold        oxygenated components of compounds 1-4 by HPLC (Exp) and theory (Calc)                  Peak I                                                                              Peak II                                                                             Peak Ill                                                                              Peak IV                                         Compound                                                                            NMR Calc                                                                             Exp                                                                              Calc                                                                             Exp                                                                              Calc                                                                              Exp Calc  Exp                                       __________________________________________________________________________    1     99.3                                                                              83.3                                                                             83.7                                                                             15.3                                                                             14.1                                                                              1.35                                                                             1.9  .07  0.3                                       1     98.3                                                                              64.0                                                                             64.5                                                                             28.8                                                                             29.0                                                                             6.2 5.6 0.9   0.8                                       1     98.0                                                                              59.1                                                                             58.1                                                                             31.4                                                                             29.4                                                                             8.0 9.8 1.3   2.7                                       2     99.6                                                                              92.3                                                                             90.0                                                                              7.4                                                                              7.9                                                                             0.3 <2  <<0.1 <0.5                                      2     97.3                                                                              57.8                                                                             52.4                                                                             32.1                                                                             33.5                                                                             8.5 11.3                                                                              1.4   2.8                                       3     99.7                                                                              94.4                                                                             93.8                                                                              5.4                                                                              5.9                                                                             <0.2                                                                              <0.5                                                                              <0.1  <0.5                                      4     99.7                                                                              94.4                                                                             93.0                                                                              5.4                                                                              7.0                                                                             <0.2                                                                              <0.5                                                                              <0.1  <0.5                                      __________________________________________________________________________

EXAMPLE 2

The same separation of compound 1 was carried out as describe in Example1, with the exception that the anion exchanger was silica gelderivatized with diethylaminoethyl cation (Nucleu-Sil, Machery & Nagel,Germany). The results are shown in the chromatogram of FIG. 2. Thehighest peak on the right corresponds to fully thioatedoligonucleotides.

EXAMPLE 3

Compound 2 of Example 1 was purified as described in Example 1 with thefollowing exceptions: (i) Buffers A and B were employed (A consisting of10 mM NaOH pH 12, and B consisting of 1.5M KBr in 10 mM NaOH ph 12.2),(ii) the PL-SAX support was loaded into a 15×0.46 cm i.d. column, and(iii) separation was achieved with a linear gradient of increasingbromide concentration by changing column conditions from buffer ratioA:B=70:30 to A:B=10:90 in 40 minutes with a flow rate of 1.0 ml/min atambient temperature. No organic modifier was added. Eluted material wasdetected by absorption at 260 nm. Results are shown in FIG. 3. It isreadily seen that the extent of separation achieved under conditions ofhigh pH and no organic modifier is almost identical to that achieved forthe identical compound under conditions of close-to-neutral pH (6.7-8.2)and at least 20% organic modifier (FIG. 1B).

What is claimed is:
 1. A method of separating completely sulfurizedphosphorothioate or phosphorodithioate oligonucleotides from a mixtureof completely sulfurized and incompletely sulfurized phosphorothioate orphosphorodithioate oligonucleotides, the method comprising the stepsof:(a) impregnating a anion exchanger with the mixture of completelysulfurized and incompletely sulfurized phosphorothioate orphosphorodithioate oligonucleotides; (b) passing through the anionexchanger a buffer solution comprising a concentration of a soft-basecounter ion which monotonically increases with time to asample-desorbing concentration; and (c) recovering the eluate from step(b).
 2. The method of claim 1 wherein said anion exchanger comprises amatrix selected from the group consisting of agarose,styrene-divinylbenzene copolymer, silica gel, and methacrylic polymer.3. The method of claim 2 wherein said matrix is derivatized with aternary or a quaternary alkylammonium cation.
 4. The method of claim 3wherein said soft-base counter ion is selected from the group consistingof bromide, thiocyanate, iodide azide, cyanate, thiosulfate, inorganicsulfide, and organic sulfides of the form RS⁻, wherein R is aryl having6 to 10 carbon atoms or alkyl having from 1 to 6 carbon atoms.
 5. Themethod of claim 4 wherein said ternary or quaternary alkylammoniumcation is selected from the group consisting of diethylaminoethyl,quaternary aminoethyl, and quaternary amine.
 6. The method of claim 5wherein said soft-base counter ion is selected from the group consistingof bromide, thiocyanate, and organic sulfide of the form RS⁻ wherein Ris phenyl, methyl, or ethyl.
 7. The method of claim 1 wherein saidsoft-base counter ion is selected from the group consisting of bromidethiocyanate, iodide, azide, cyanate, thiosulfate, inorganic sulfide, andorganic sulfides of the form RS⁻, wherein R is aryl having 6 to 10carbon atoms or alkyl having from 1 to 6 carbon atoms.
 8. The method ofclaim 7 wherein said soft-base counter ion is selected from the groupconsisting of bromide, thiocyanate, azide, sulfide, iodide, and organicsulfide of the form RS⁻ wherein R is phenyl, methyl, or ethyl.
 9. Themethod of claim 8 wherein said soft-base counter ion is selected fromthe group consisting of bromide, thiocyanate, azide, sulfide, andiodide.
 10. The method of claim 9 wherein said buffer solution comprisesa soft-base counter ion at a concentration which increases linearly withtime from a starting concentration to said sample-desorbingconcentration.
 11. The method of claim 10 wherein said soft-base counterion increases in concentration at a rate in the range of 1 to 2 percentper minute.
 12. A method of measuring the fraction of completelysulfurized phosphorothioate or phosphorodithioate oligonucleotides in amixture of completely sulfurized and incompletely sulfurizedphosphorothioate or phosphorodithioate oligonucleotides, the methodcomprising the steps of:(a) impregnating a anion exchanger with themixture of completely sulfurized and incompletely sulfurizedphosphorothioate or phosphorodithioate oligonucleotides; (b) passingthrough the exchanger a buffer solution comprising a concentration of asoft-base counter ion which monotonically increases with time to asample-desorbing concentration so that a chromatogram of the elutedcompletely sulfurized and incompletely sulfurized phosphorothioate andphosphorodithioate oligonucleotides is formed; and (c) computing theratio of the peak area corresponding to the completely sulfurizedphosphorothioate or phosphorodithioate oligonucleotide on thechromatogram to the total area under all peaks corresponding to bothcompletely sulfurized and incompletely sulfurized phosphorothioate orphosphorodithioate oligonucleotides on the chromatogram.
 13. The methodof claim 12 wherein said soft-base counter ion is selected from thegroup consisting of bromide, thiocyanate, azide, sulfide, and iodide.14. The method of claim 13 wherein said soft-base counter ion isselected from the group consisting of bromide and thiocyanate.
 15. Themethod of claim 14 wherein said buffer solution comprises aconcentration of a soft-base counter ion which increases linearly withtime to said sample-desorbing concentration.